Coxiella Burnetii and Rickettsiella Grylli Genome Project


The goal of this project is to generate complete genome sequence data from four Coxiella burnetii isolates and Rickettsiella grylli in order to define variations in strains that are responsible for differences in virulence and host range. Additionally, comparative genomics of a newly-discovered Coxiella sp. of ticks and Coxiella burnetii will be done. The goals of the comparative genomics project are to characterize a core set of proteins for Coxiella spp. and to identify genes unique to C. burnetii that could facilitate this pathogen's transmission to or virulence in vertebrates.


Q fever is a Category B select agent and has long been considered of high potential as an agent for biological warfare or terrorism. [2] Although infection mortality is low and secondary transmission between humans is rare, the acute illnesses frequently incapacitate infected individuals, and the attack rate can be high after point-source exposures. [3, 4] Moreover, in some cases acute illness is followed by chronic infection, such as "culture-negative" endocarditis, [3] or by a disabling post-infection fatigue syndrome. [5] Besides natural cycles of C. burnetii among wildlife and their tick vectors, the disease is enzootic and endemic in many regions of the world with cattle, goat, and sheep raising. The high concentrations of infectious microorganisms in placentas, milk, vaginal secretions, and feces of infected animals provide a ready, "low-tech" source of an infectious agent for would-be terrorists. Excreted organisms are highly resistant to drying, and the minimum inoculum for infection by the aerosol route is low. There have been several outbreaks of disease from point-source exposures through inadvertent aerosols, so sophisticated "weaponization" is not a requirement for successful delivery of this agent. Ampicillin-resistant C. burnetii have been obtained through genetic transformation with C. burnetii-E. coli shuttle plasmids, [5] so engineering for resistance to commonly recommended antibiotics, such as tetracyclines, is feasible.

Strain Selection

Diverse geographical distribution of C. burnetii has resulted in the accumulation of varied isolates that appear to be serologically similar. Isolates from mammalian and arthropod hosts have been differentiated based on genetic and phenotypic differences into at least 6 different genomic groups. [26-29] We propose to have a representative strain from each genomic group sequenced. The strains selected constitute a selection based on the virulence potential, disease phenotypes, host organism, geographical distribution, and gross genomic distinctions.

Biology of C. burnetti

A hallmark property of C. burnetii is its ability to remain infectious after extended exposure to extreme environmental circumstances (e.g. elevated temperature, desiccation, osmotic shock, ultraviolet light, and chemical disinfectants) [6]. This ability to survive a harsh intra- and extracellular environment may rely, in part, on a developmental life cycle with various forms specialized for propagation, surviving intracellular stress and persisting in the extracellular environment (in a non-replicating state) for long periods between hosts [9] [6].

In vitro, C. burnetii passively enters and replicates within a variety of epithelial, fibroblast, and macrophage-like cell lines [10]. Internalization into host cells occurs by a microfilamentdependent parasite-directed endocytic process [11]. The nascent parasite-containing phagosomes mature through the endocytic pathway, eventually acquiring the properties of secondary lysosomes. C. burnetii has an absolute requirement for a moderately acidic pH to activate metabolism and exploits the only intracellular niche capable of establishing such a pH [7, 8, 12]. The organism replicates to high numbers, albeit at a slow replication rate (~12 h doubling time) within this vacuole, despite the presence of toxic host factors, such as acid hydrolase, oxygen and nitrogen radicals, and defensins which are normally considered bactericidal [10].

C. burnetii is generally maintained in nature by cycles involving many species of animals, both herbivores and carnivores, as well as arthropods which become infected by taking a blood meal [13] [14]. Domestic animals are involved in the cycle of infection (cattle, sheep and goats are the primary reservoirs) and humans become infected via aerosols that originate primarily from contaminated animal products [15]. These infectious cycles provide ecological niches that might select for highly virulent genetic variants. Isolates obtained from a variety of wild vertebrates and arthropods have been classified into genomic groups by various methods and while these groups do not correlate with host, geographical location or date of isolation, the type of human disease caused by isolates in each group is similar, implying that specific isolate groups have a particular virulence potential in humans, determined by specific factors and molecular mechanisms encoded by each.

C. burnetii causes a wide spectrum of human disease. The disease manifestations of C. burnetii infection in humans can be separated into acute and chronic illnesses. Acute disease commonly presents as a flu-like illness with hallmark cyclic fever and periorbital headache [16]. Pneumonitis and hepatitis are frequent complications but acute disease is commonly self-limiting. Various antibiotics, including tetracyclines, are effective for abrogating acute disease [17]. Until recently, clinically, the illness fell within the group of FUO (fever of unknown origin) syndromes and was not commonly recognized or diagnosed. In many areas of the world, a high percentage of the population (10-20%) has serological evidence of previous infection [18]. In contrast, chronic infection is much less common and has a grim prognosis [19] [20]. Chronic disease most frequently manifests as endocarditis and hepatitis with recognition of these infections increasing worldwide. Recently a group of chronic infection patients, reported in Australia, presented with similar symptoms to chronic fatigue syndrome [21]. Chronic infections appear to be associated with a suppression of the cell-mediated immune system [22]. Chronic infections have not responded well to a variety of antibiotic regimens, with patients receiving a combination of doxycycline plus chloroquine, administered over 1-2 years, showing the best outcome [23]. Thus acute infection with C. burnetii leads to long-term vascular inflammation and points to latent infections comparable to that noted for Chlamydia pneumonia and other infectious agents. From a public health and bio-defense perspective, exposure to different strains, either natural, or due to illegitimate release, may have quite different outcomes. Exposure to acute disease isolates would likely result in predominantly flu-like illness with the potential for long term cardiovascular disease risk. Exposure of a population to chronic disease isolates would probably not result in significant immediate disease cases, but would likely result in chronic illnesses developing months or years from initial exposure.

Biology of R. grylli

Rickettsiella grylli is an obligate intracellular parasite of Gryllus bimaculatus, and related species of crickets. Members of the genus Rickettsiella are intracellular pathogens of invertebrates, including insects, crustaceans, and arachnids [24, 25]. The infectious forms are Gram-negative rod-shaped organisms that develop intracelluarly into larger vegetative forms, similar to the developmental cycle observed for Chlamydia spp and Coxiella. Bacterial replication takes place in cell vacuoles in the fat body, the hepatopancreas, and other organs of invertebrate hosts. Growth in cell-free media has not been demonstrated. They are pathogenic for their larval hosts and young and mature stages of invertebrate hosts, but of little virulence for vertebrates. The chief interest in Rickettsiellae stems from their infection of lab insectaries and other animal collections, as well as biocontrol for agricultural pests. They are maintained in the soil for years, and infection arises from contaminated soil rather than transovarian passage. The wide distribution of Rickettsiella geographically and in arthropod taxa suggests an early appearance in the course of evolution.

Since studies of host specificity, cultivation or antigenic analyses are not extensive, most of the knowledge is based on light and EM observations. The developmental cycle is comprised of a small, dense infectious particle, which is phagocytosed by the host cell, and within the intravacuolar compartment, it differentiates into less electron-dense intermediate- and large-sized forms that multiply. As the vacuole fills to capacity, large forms recondense to smaller dense particles that eventually escape the vacuole to start another infectious cycle. This developmental cycle is very similar to that described for C. burnetii involving 2-3 distinct morphological variants.

Virulence for invertebrate hosts other than host of isolation has been studied. R. grylli has been found to grow in Orthoptera, Lepidoptera and Coleoptera orders of insects, and Isopod and Amphipod crustaceans. R. grylli has also been shown to multiply in the mouse when large doses are inoculated by the intraperitoneal route or by inhalation, but the mouse overcomes infection and organisms are cleared in about 2 weeks.


Coxiella burnetii MSU Goat Q1

This strain, a goat abortion isolate, is part of an isolate group that has a distinct plasmid type, QpRS. Virulence studies in a guinea pig model suggest an intermediate phenotype for this strain including low fever and no death in an aerosol challenge experiment compared to highly virulent acute isolate groups.

  Status Description Date
Annotation Published On contigs: AAUP01000001 : AAUP01000150 Oct 24 2007
Assembly Previous assembly AI 2992 Jan 29 2007
Assembly Published AI 2993 Sep 20 2007
Sequence Previous WGS AAUP01000000 Nov 16 2006
Sequence WGS Published AAUP02000000 Sep 7 2007
Taxonomy Available Taxonomy ID 360116  

Coxiella burnetii Dugway 7E9-12

The Dugway strain is a member of an isolate group that contains a distinct plasmid, QpDG.

  Status Description Date
Annotation Published On genome records: CP000735.1, CP000735.1 Aug 1 2007
Assembly Published AI 2954 Aug 20 2007
Assembly Previously assembly AI 1210 May 3 2006
Sequence Previous WGS AAQI01000000 Apr 27 2006
Sequence Genome Published CP000733.1, CP000735.1 Aug 1 2007
Taxonomy Available Taxonomy ID 382253  
Trace Archive Available 52,333 traces  

Coxiella burnetii RSA 331

This strain is part of an isolate group that is primarily associated with acute Q-fever. Henzerling strain RSA 331 was isolated from blood of an infected patient in Italy in 1945.

  Status Description Date
Annotation Published On genome records: CP000889.1, CP000890.1 Dec 5 2007
Assembly Published AI 3451 Jan 7 2008
Assembly Previous Assembly AI 1259 May 18 2006
Assembly Previous Assembly AI 2992 Sep 20 2007
Sequence Previous WGS AAQO02000000 Aug 20 2007
Sequence Genome Published CP000889.1, CP000890.1 Dec 5 2007
Sequence   AAQO01000000 May 12 2006
Taxonomy Available Taxonomy ID 360115  
Trace Archive Available 26,017 traces  

Coxiella burnetii RSA 334

  Status Description Date
Annotation Published On contigs: AAYJ00000001 : AAYJ00000148 Jan 17 2008
Assembly Published AI 2674 May 1 2007
Sequence WGS Published AAYJ01000000 Mar 19 2007
Taxonomy Available Taxonomy ID 360117  
Trace Archive Available 25,865 traces  

Rickettsiella grylli

  Status Description Date
Annotation Published On contigs: AAQJ02000001 : AAQJ02000002 Nov 2 2007
Assembly Published AI 3388 Dec 12 2007
Assembly Previous assembly AI 1211 May 3 2006
Sequence Previous WGS AAQJ01000000 Apr 28 2006
Sequence WGS Published AAQJ02000000 Nov 5 2007
Taxonomy Available Taxonomy ID 59196  
Trace Archive Available 26,343 traces  

Production Status

Organism Name Status Taxon Trace Sequence Assembly Annotation
Coxiella burnetii Dugway 7E9-12 Complete X T G A O
Coxiella burnetii MSU Goat Q177 Complete X   W A O
Coxiella burnetii RSA 331 Complete X T G A O
Coxiella burnetii RSA 334 Complete X T W A O
Rickettsiella grylli Complete X T W A O


  1. Seshadri, R., I. T. Paulsen, J. A. Eisen, T. D. Read, K. E. Nelson, W. C. Nelson, N. L. Ward, H. Tettelin, T. M. Davidsen, M. J. Beanan, R. T. Deboy, S. C. Daugherty, L. M. Brinkac, R., A. Thompson, J. E. Samuel, C. M. Fraser, and J. F. Heidelberg. 2003. Complete genome sequence of the Q-fever pathogen Coxiella burnetii. Proc Natl Acad Sci U S A 100:5455-60.
  2. Madariaga, M. G., K. Rezai, G. M. Trenholme, and R. A. Weinstein. 2003. Q fever: a biological weapon in your backyard. Lancet Infect Dis 3:709-21.
  3. Raoult, D., T. Marrie, and J. Mege. 2005. Natural history and pathophysiology of Q fever. Lancet Infect Dis 5:219-26.
  4. Thompson, H. A., D. T. Dennis, and G. A. Dasch. 2005. Q Fever, p. 328-342. In J. L. Goodman, D. T. Dennis, and D. E. Sonenshine (ed.), Tick-Borne Diseases of Humans. ASM Press, Washington, D. C.
  5. Ayres, J. G., N. Flint, E. G. Smith, W. S. Tunnicliffe, T. J. Fletcher, K. Hammond, D. Ward, and B. P. Marmion. 1998. Post-infection fatigue syndrome following Q fever. Qjm 91:105-23.
  6. Heinzen, R.A., T. Hackstadt, and J.E. Samuel, Developmental biology of Coxiella burnettii. Trends Microbiol, 1999. 7(4): p. 149-54.
  7. Akporiaye, E.T., et al., Lysosomal response of a murine macrophage-like cell line persistently infected with Coxiella burnetii. Infect Immun, 1983. 40(3): p. 1155-62.
  8. Hackstadt, T. and J.C. Williams, Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii. Proc Natl Acad Sci U S A, 1981. 78(5): p. 3240-4.
  9. McCaul, T.F., N. Banerjee-Bhatnagar, and J.C. Williams, Antigenic differences between Coxiella burnetii cells revealed by postembedding immunoelectron microscopy and immunoblotting. Infect Immun, 1991. 59(9): p. 3243-53.
  10. Baca, O.G. and D. Paretsky, Q fever and Coxiella burnetii: a model for host-parasite interactions. Microbiol Rev, 1983. 47(2): p. 127-49.
  11. Baca, O.G., D.A. Klassen, and A.S. Aragon, Entry of Coxiella burnetii into host cells. Acta Virol, 1993. 37(2-3): p. 143-55.
  12. Heinzen, R.A., et al., Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis. Infect Immun, 1996. 64(3): p. 796-809.
  13. Marrie, T.J., Epidemiology of Q fever , in Q fever, The Disease , T.J. Marrie, Editor. 1990, CRC Press: Boca Raton, Fla. p. 49-70.
  14. Kaplan, M.M. and P. Bertagna, The geographical distribution of Q fever. Bull World Health Organ, 1955. 13(5): p. 829-60.
  15. Babudieri, C., Q fever: a zoonosis. Adv Vet. Sci., 1959. 5: p. 81-84.
  16. Raoult, D. and T. Marrie, Q fever. Clin Infect Dis, 1995. 20(3): p. 489-95; quiz 496.
  17. Raoult, D., Treatment of Q fever. Antimicrob Agents Chemother, 1993. 37(9): p. 1733-6.
  18. Htwe, K.K., et al., Prevalence of antibodies to Coxiella burnetii in Japan. J Clin Microbiol, 1993. 31(3): p. 722-3.
  19. Sidwell, R.W., B.D. Thorpe, and L.P. Gebhardt, Studies of Latent Q Fever Infections. Ii. Effects of Multiple Cortisone Injections. Am J Hyg, 1964. 79: p. 320-7.
  20. Brouqui, P., et al., Chronic Q fever. Ninety-two cases from France, including 27 cases without endocarditis. Arch Intern Med, 1993. 153(5): p. 642-8.
  21. Marmion, B.P., et al., Protracted debility and fatigue after acute Q fever. Lancet, 1996. 347(9006): p. 977-8.
  22. Raoult, D., Host factors in the severity of Q fever. Ann N Y Acad Sci, 1990. 590: p. 33-8.
  23. Jabarit-Aldighieri, N., H. Torres, and D. Raoult, Susceptibility of Rickettsia conorii, R. rickettsii, and Coxiella burnetii to PD 127,391, PD 131,628, pefloxacin, ofloxacin, and ciprofloxacin. Antimicrob Agents Chemother, 1992. 36(11): p. 2529-32.
  24. Weiss, E., G.A. Dasch, and K. Chang, Rickettsiella , in Bergey's Manual of Systematic Bacteriology , N.R. Krieg, Editor. 1984, Williams and Wilkins: Baltimore, MD. p. 713-717.
  25. Vago, C. and R. Martoja, [Rickettsiosis in the Gryllidae (Orthoptera)]. C R Hebd Seances Acad Sci, 1963. 256: p. 1045-7.
  26. Heinzen, R., et al., Use of pulsed field gel electrophoresis to differentiate Coxiella burnetii strains. Ann N Y Acad Sci, 1990. 590: p. 504-13.
  27. Vodkin, M.H., J.C. Williams, and E.H. Stephenson, Genetic heterogeneity among isolates of Coxiella burnetii. J Gen Microbiol, 1986. 132 ( Pt 2): p. 455-63.
  28. Hendrix, L.R., J.E. Samuel, and L.P. Mallavia, Differentiation of Coxiella burnetii isolates by analysis of restriction-endonuclease-digested DNA separated by SDS-PAGE. J Gen Microbiol, 1991. 137 ( Pt 2): p. 269-76.
  29. Jager, C., et al., Molecular characterization of Coxiella burnetii isolates. Epidemiol Infect, 1998. 120(2): p. 157-64.


John Heidelberg
University of Southern California

Ramana Madupu
J. Craig Venter Institute

Garry Myers
Institute for Genome Sciences, University of Maryland School of Medicine

James Samuel
Texas A&M Health Science Center

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